Cancer stem cells (CSC) are now recognized as the main cause for initiation, promotion and progression of most of the cancers, including colorectal cancer (CRC). Despite this fact, efficacy of chemopreventive agents towards CSC generation leading to cancer initiation and tumorigenesis has not yet been well- defined. Our completed and published studies show strong preventive and therapeutic efficacy of silibinin against CRC xenografts in nude mice, AOM-induced colon tumorigenesis in A/J mice and spontaneous tumorigenesis in APCmin/+ models, together with strong anti-proliferative, pro-apoptotic and anti-inflammatory effects. Also, silibinin strongly decreases the expression of NOTCH survival signaling molecules and b- catenin together with an inhibition in b-catenin transcriptional activity, which are important regulators of CSC renewal; as well as decreases the expression of CD44, an important colon CSC marker. Another important observation we made is that silibinin significantly decreases colonosphere formation (a stem cell characteristic) by CRC cells, thereby highlighting its potential to target colon CSC. Present continuation application builds upon these highly novel observations, where we propose to examine and establish silibinin efficacy on colon CSC generation, expansion, self-renewal and differentiation in the context of CRC chemoprevention. The central hypothesis proposed is that silibinin targets CSC generation and expansion by modulating signaling pathways as well as pro-inflammatory milieu that regulate CSC 'self renewal' and 'differentiation' capabilities, leading to its strong efficacy against CRC initiation, promotion and recurrence. Specific aims are: I) to examine and establish silibinin efficacy on colon CSC generation and expansion, and associated signaling pathways; II) to examine and establish silibinin efficacy on self-renewal and differentiation potential of colon CSC; and III) to examine and define silibinin efficacy on the inflammatory milieu of the colon CSC 'niche'. We believe that the present proposal would have profound impact as 'first of its kind study' with a goal to prevent CRC by targeting 'cancer stem cells'. We rationalized our approach based upon the fact that stem cells are the long lived population in colon and are the preferential target for initial oncogenic mutations, and therefore, CSC are identified as the real driving force behind CRC initiation, promotion and progression. We strongly believe that present proposal will provide a new direction to 'cancer chemoprevention research' by laying greater focus on the role of CSC in prevention and recurrence of disease. Human Health Relevance: Since silibinin consumption is safe in humans and its bioavailability has already been established in CRC patients, the outcomes of the studies in present proposal would have significant clinical impact on the rationalized use of silibinin in human CRC control.